Specialized receptor neurons for pheromones and host plant odors in the boll weevil, Anthonomus grandis Boh. (Coleoptera: Curculionidae)

Antennal olfactory receptor neurons in the boll weevil, Anthonomusgrandis, were investigated through single neuron recordings.Receptor neurons for both pheromone components and host plantodors were associated with type I sensilla within the sensoryband regions. Nine types of receptor neurons were identified,based on their responsiveness to the four aggregation pheromonecomponents and selected host plant odors. Three receptor neurontypes responded to either compound I, II or IV of the aggregationpheromone. Dose—response curves were similar for eachof these receptor neuron types, which differed only in theirkey compound. In each instance, I neurons responded primarilyto (+)-I, the optical isomer produced by the boll weevil whichwas found to be active in field tests. Receptor neurons forII also responded to a lesser degree to III, its aldehydic analog,at the same stimulus load. Six additional receptor neuron typesresponded to selected host plant odors: ß-caryophyllene,trans-2-hexen-l-o1 and other six carbon alcohols and aldehydes,trans-ß-ocimene, benzaldehyde, linalool, and B-bisabolol.These neurons were as responsive as, or in some cases more responsiveat the same stimulus load as receptor neurons for pheromonecomponents. Receptor neurons responsive to six-carbon alcoholsand aldehydes were generally most responsive to trans-2-hexen-l-ol.Receptor neurons for other plant odors responded principallyto only one compound among the odorants tested. However, responsesof these neurons were not uniform, suggesting possible specializationfor other unidentified key odorants. Comparisons were also madebetween single neuron and electroantennogram responses. Theresults indicate that the boll weevil, a narrowly oligaphagousinsect, detects its host plant at some distance, and utilizesinformation about a wide range of chemical structures in itsolfactory-mediated behavior.     

Olfactory receptor neurons in two Heliothine moth species responding selectively to aliphatic green leaf volatiles, aromatic compounds, monoterpenes and sesquiterpenes of plant origin

Moths of the subfamily Heliothinae are suitable models for comparative studies of plant odour information encoded by the olfactory system. Here we identify and functionally classify types of olfactory receptor neurons by means of electrophysiological recordings from single receptor neurons linked to gas chromatography and to mass spectrometry. The molecular receptive ranges of 14 types in the two polyphagous species Heliothis virescens and Helicoverpa armigera are presented. The receptor neurons are characterized by a narrow tuning, showing the best response to one primary odorant and weak responses to a few chemically related compounds. The most frequently occurring of the 14 types constituted the receptor neurons tuned to (+)-linalool, the enantioselectivity of which was shown by testing two samples with opposite enantiomeric ratios. These neurons, also responding to dihydrolinalool, were found to be functionally similar in the two related species. The primary odorants for 10 other receptor neuron types were identified as (3Z)-hexenyl acetate, (+)-3-carene, trans-pinocarveol, trans-verbenol, vinylbenzaldehyde, 2-phenylethanol, methyl benzoate, alpha-caryophyllene and caryophyllene oxide, respectively. Most odorants were present in several host and non-host plant species, often in trace amounts. The specificity as well as the co-localization of particular neuron types so far recorded in both species showed similarities of the olfactory systems receiving plant odour information in these two species of heliothine moths.     

Receptor neuron discrimination of the germacrene D enantiomers in the moth Helicoverpa armigera

Plants release complex mixtures of volatiles, including chiral constituents. In the search for the biologically relevant plant odorants, gas chromatography linked to electrophysiological recordings from single receptor neurons has been employed. In heliothine moths, including the females of the Eurasian cotton bollworm moth Helicoverpa armigera, a major type of receptor neurons is identified, showing high sensitivity and selectivity for the sesquiterpene germacrene D. In the present study, gas chromatography with a chiral column linked to single cell recordings were performed. It was found that all germacrene D neurons belonged to one type; all responded to both enantiomers, but (-)-germacrene D had approximately 10 times stronger effect than (+)-germacrene D. Parallel dose-response curves for the two enantiomers were obtained by direct stimulations. The enantiomeric composition of germacrene D, which differed in six plant species and in different individuals of one species, was determined on the basis of the neuron responses. The results, showing the presence of one neuron type for receiving the information about germacrene D in the various plants, suggests that the two enantiomers mediate the same kind of information to the moth, but with different intensity.     

Encoding of plant odours by receptor neurons in the pine weevil (Hylobius abietis) studied by linked gas chromatography-electrophysiology

Receptor neuron responses to plant volatiles, trapped by head-space procedures, were examined in the pine weevil Hylobius abietis, using gas chromatography linked with electrophysiological recordings from single neurons. Seventy-two receptor neurons were tested 173 times for various plant volatile mixtures, either via a polar or a non-polar column.

1. All responses appeared as increased firing rates which followed the concentration profiles of the GC-eluted compounds.
2. The neurons were classified separately for the two column types in 17 and 19 groups respectively, according to the compounds they responded to. It suggests that the plant odour information is encoded by a large, but limited number of receptor neuron types.
3. Most neurons responded to a limited number of compounds (1–5) and showed a marked best response to one of them, whereas additional responses to several other components which seems to be structurally similar, was recorded for some neurons. It suggests that the plant odour receptor neurons are rather narrowly than broadly tuned, and that each neuron is specialized for receiving information about one or a few related compounds.
4. Most neurons responded to monoterpenes, whereas the other neurons responded to compounds of other categories.
5. Both major and minor plant volatile components activated specifically receptor neurons.

     

Discrimination between enantiomers of linalool by olfactory receptor neurons in the cabbage moth Mamestra brassicae (L.)

Plants emit complex blends of volatiles, including chiral compounds that might be detected by vertebrates and invertebrates. Insects are ideal model organisms for studying the underlying receptor neuron mechanisms involved in olfactory discrimination of enantiomers. In the present study, we have employed two-column gas chromatography linked to recordings from single olfactory receptor neurons of Mamestra brassicae, in which separation of volatiles in a polar and a chiral column was performed. We here present the response properties of olfactory receptor neurons tuned to linalool. The narrow tuning of these receptor neurons was demonstrated by their strong responses to (R)-(-)-linalool, the weaker responses to the (+)-enantiomer as well as a few structurally related compounds, and no responses to the other numerous plant released volatiles. The enantioselectivity was verified by parallel dose-response curves, that of (R)-(-)-linalool shifted 1 log unit to the left of the (S)-(+)-linalool curve. A complete overlap of the temporal response pattern was found when comparing the responses of the same strength. Analysis of the spike amplitude and waveform indicated that the responses to the two enantiomers originated from the same neuron.     

The plant sesquiterpene germacrene D specifically activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens

Plants release hundreds of volatiles that are important in interactions with insects or other organisms. However, knowledge is scarce as to which of the compounds are detected by the organism's olfactory receptor neurons. In the present study, single receptor neurons on the antennae of the tobacco budworm moth, Heliothis virescens, were screened for their sensitivities to naturally produced plant volatiles by the use of gas chromatography linked to electrophysiological recordings from single cells (GC-SCR). Plant volatiles, collected by aeration of host and non-host plants, were tested on each receptor neuron via parallel GC-columns. Thus, simultaneous recordings of the gas chromatogram and the neuron responses to each component were obtained. One type of receptor neuron, appearing in 80% of all experiments, responded with high sensitivity and selectivity to one particular component, present in host as well as non-host mixtures. The component, identified as a sesquiterpene hydrocarbon by linked gas chromatography-mass spectrometry, was isolated from a sesquiterpene fraction of cubebe oil and identified by NMR as germacrene D. The purified compound was then re-tested via gas chromatography on the same receptor neuron type, verifying the identification. A weaker response to another sesquiterpene hydrocarbon was also recorded.     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Plant volatiles activating specific olfactory receptor neurons of the cabbage moth Mamestra brassicae L. (Lepidoptera, Noctuidae)

Herbivore insects are suitable model organisms for studying how plant odor information is encoded in olfactory receptor neurons (RNs). By the use of gas chromatography linked to electrophysiological recordings from single RNs, screening for sensitivity to naturally produced plant odorants is possible in order to determine the molecular receptive ranges of the neurons. Using this method, we have in this study of the cabbage moth, Mamestra brassicae, classified 21 types of olfactory RNs according to their responses to odorants present in the host plants of Brassicae, in the related species of Arabidopsis, as well as in essential oils of nonhost plants like ylang-ylang. Most of the RNs were tuned to one or a few structurally similar compounds, showing minimal overlap of their molecular receptive ranges. Whereas some RNs displayed a novel tuning, others were tuned to the same compounds as neurons in other insect species. We also found colocation in the same sensillum of 3 RN types with the same response characteristics and tuning as 3 colocated types described in heliothine moths living on different host plants. The presence of similar RN types across different insect species implies conservation or reappearance of the RN types, independent of the evolution of host plant ranges.     

Olfactory receptor antagonism between odorants

The detection of thousands of volatile odorants is mediated by several hundreds of different G protein-coupled olfactory receptors (ORs). The main strategy in encoding odorant identities is a combinatorial receptor code scheme in that different odorants are recognized by different sets of ORs. Despite increasing information on agonist-OR combinations, little is known about the antagonism of ORs in the mammalian olfactory system. Here we show that odorants inhibit odorant responses of OR(s), evidence of antagonism between odorants at the receptor level. The antagonism was demonstrated in a heterologous OR-expression system and in single olfactory neurons that expressed a given OR, and was also visualized at the level of the olfactory epithelium. Dual functions of odorants as an agonist and an antagonist to ORs indicate a new aspect in the receptor code determination for odorant mixtures that often give rise to novel perceptual qualities that are not present in each component. The current study also provides insight into strategies to modulate perceived odorant quality.     

Identification of plant odours activating receptor neurones in the weevil<Emphasis Type="Italic"> Pissodes notatus</Emphasis> F. (Coleoptera,Curculionidae)

Plants release complex mixtures of volatiles important in the interaction with insects and other organisms. In the search for compounds that contribute to the perception of odour quality in the weevil Pissodes notatus, single olfactory receptor neurones on the antennae were screened for sensitivity to naturally produced plant volatiles by the use of gas chromatography linked to single cell recordings. We here present 60 olfactory neurones responding to 25 of the numerous compounds released by host and non-host plants. All the neurones show high selectivity and are classified into 12 distinct types. The two most abundant types respond to alpha-pinene, beta-pinene, and 3-carene ( n=17), and to isopinocamphone and pinocamphone ( n=17), respectively. Other neurone types respond to limonene ( n=9), beta-phellandrene ( n=3), and fenchone ( n=4). Responses to beta-caryophyllene ( n=1) and to ethanol ( n=4) are also shown. Except for two pairs, the neurone types do not show overlap of the molecular receptive range. The active compounds are present in the host, Pinus pinaster, as well as in non-hosts, supporting the idea that plant odour quality is mediated by the ratio of the compounds rather than specific odorants.     

Responses to sex pheromone and plant odours by olfactory receptor neurons housed in sensilla auricillica of the codling moth, Cydia pomonella (Lepidoptera: Tortricidae)

Antennal olfactory receptor neurons located in a limited number of two types of sensilla auricillica, the rabbit-eared shoehorn and the regular shoehorn, located on the 5-30 flagellomere of the codling moth, Cydia pomonella, antenna were screened for selectivity to 11 plant compounds, the major sex pheromone component, three minor pheromone components and one behavioural antagonist. Both types of sensilla housed at least three neurons characterised by different action potential amplitudes. Neurons in both males and females responded to the plant compounds, ethyl (E,Z)-2,4-decadienoate, (+/-)-linalool, (E)-ss-farnesene, hexanol, (Z)-3-hexenyl acetate, 4,8-dimethyl-1,3,(E)7-nonatriene, nonanol, the major pheromone component codlemone [(E,E)-8,10-dodecadienol] and the minor pheromone component tetradecanol. Additionally, (E,E)-alpha-farnesene and (Z)-3-hexenol elicited responses specifically in female neurons, whereas (E,E)-farnesol elicited a specific response in a male neuron. Neurons responded to 1-3 odorants, with sometimes overlapping response spectra. A scanning electron microscopic study of the antennae of both sexes supported an earlier study, apart from that long s. trichodea were present in a wreath at the proximal margin of the flagellomere and in addition evenly distributed over the remaining surface, and a previously non-described sensillum type with external basiconic features was revealed, distributed on the proximal and medial region of the flagellomeres.     

Methyl salicylate, identified as primary odorant of a specific receptor neuron type, inhibits oviposition by the moth Mamestra brassicae L. (Lepidoptera, noctuidae)

The cabbage moth, Mamestra brassicae L. (Lepidoptera, Noctuidae), is a polyphagous species that is often choosing plants of Brassica as hosts for oviposition. In the search for biologically relevant odorants used by these moths, gas chromatography linked to electrophysiological recordings from single receptor neurons (RNs) has been employed, resulting in classification of distinct types of neurons. This study presents specific olfactory RNs responding to methyl salicylate (MeS) as primary odorant and showing a weak response to methyl benzoate, the 2 aromatic compounds occurring together in several plant species. In 2 cases, the neuron was colocated with another RN type responding to 6 green leaf volatiles: 1-hexanol, (3Z)-hexen-1-ol, (2E)-hexen-1-ol, (3Z)-hexenyl acetate, (2Z)-hexen-1-ol, and an unidentified compound. Whereas the specific RNs detected the minor amounts of MeS in some plants, the compound was not found by gas chromatography linked to mass spectrometry in intact plants, but it was found after herbivore attack. The behavioral effect of MeS was studied in outdoor test arenas with Brassica napus and artificial plants. These experiments indicated that mated M. brassicae females avoid plants with dispensers emitting MeS. As it is induced by caterpillar feeding, this compound may mediate a message to mated M. brassicae females that the plant is already occupied.     

Antennal lobe projection destinations of Helicoverpa zea male olfactory receptor neurons responsive to heliothine sex pheromone components

We used single sensillum recordings to define male Helicoverpa zea olfactory receptor neuron physiology followed by cobalt staining to trace the axons to destination glomeruli of the antennal lobe. Receptor neurons in type A sensilla that respond to the major pheromone component, (Z)-11-hexadecenal, projected axons to the cumulus of the macroglomerular complex (MGC). In approximately 40% of these sensilla a second receptor neuron was stained that projected consistently to a specific glomerulus residing in a previously unrecognized glomerular complex with six other glomeruli stationed immediately posterior to the MGC. Cobalt staining corroborated by calcium imaging showed that receptor neurons in type C sensilla sensitive to (Z)-9-hexadecenal projected to the dorsomedial posterior glomerulus of the MGC, whereas the co-compartmentalized antagonist-sensitive neurons projected to the dorsomedial anterior glomerulus. We also discovered that the olfactory receptor neurons in type B sensilla exhibit the same axonal projections as those in type C sensilla. Thus, it seems that type B sensilla are anatomically type C with regard to the projection destinations of the two receptor neurons, but physiologically one of the receptor neurons is now unresponsive to everything except (Z)-9-tetradecenal, and the other responds to none of the pheromone-related odorants tested.     

Searching for the Ligands of Odorant Receptors

Through the sense of smell mammals can detect and discriminate between a large variety of odorants present in the surrounding environment. Odorants bind to a large repertoire of odorant receptors located in the cilia of olfactory sensory neurons of the nose. Each olfactory neuron expresses one single type of odorant receptor, and neurons expressing the same type of receptor project their axons to one or a few glomeruli in the olfactory bulb, creating a map of odorant receptor inputs. The information is then passed on to other regions of the brain, leading to odorant perception. To understand how the olfactory system discriminates between odorants, it is necessary to determine the odorant specificities of individual odorant receptors. These studies are complicated by the extremely large size of the odorant receptor family and by the poor functional expression of these receptors in heterologous cells. This article provides an overview of the methods that are currently being used to investigate odorant receptor–ligand interactions.     

Detection of fruit- and flower-emitted volatiles by olfactory receptor neurons in the polyphagous fruit chafer Pachnoda marginata (Coleoptera: Cetoniinae)

Olfactory receptor neurons on the antennae of the African fruit chafer species Pachnoda marginata (Coleoptera: Scarabaeidae) were examined through extensive use of gas chromatography linked with electrophysiological recordings from single olfactory receptor neurons. Contacted neurons were stimulated with a large number of extracted volatiles from 22 different fruits and with 64 synthetic plant compounds. Extracted fruit volatiles were identified using linked gas chromatography-mass spectrometry. In total, 48 different odor compounds were found to elicit responses. Analysis of the response spectra of the contacted neurons (n = 232) revealed the presence of 28 classes of receptor neurons. The neurons exhibited strong selectivity as well as high sensitivity. Eleven of the identified classes were selectively activated by single compounds, while the remaining were activated by 2-6 compounds. Several receptor neurons that were activated by more than one compound responded to compounds sharing basic structural similarities. The results support the growing hypothesis that a significant proportion of plant-odor receptor neurons in insects are highly sensitive and selective for single odors.     

A comparative study of pheromone perception in two species of Noctuid moths

The electrical activity of single olfactory receptor neurons in male soybean looper (SBL) Pseudoplusia includens(Walker) and cabbage looper (CL) Trihoplusia ni(Hübner) moths was evaluated in response to stimulation with fixed amounts of the individual components of their respective pheromone blends. In common with earlier observations in the CL, there are at least two classes of morphologically distinct pheromone sensitive sensilla on the antenna of male SBL, each of which contains two olfactory receptor neurons. In both species, one class of sensilla contains an olfactory receptor neuron sensitive to (Z)-7-dodecen-1-ol acetate (Z-7, 12:AC), the major component in each insect's blend, and a companion receptor neuron which is sensitive to (Z)-7-dodecen-1-ol (Z7,12: OH). In both species the second class of sensilla contains an olfactory receptor neuron which is sensitive to one of the minor components of the pheromone blend. (Z)-5-dodecen-1-ol acetate (Z-5,12:AC) is an effective stimulus in SBL, whereas (Z)-7-tetradecen-1-ol acetate (Z-7,14:AC) is an effective stimulus in CL. However, these two stimulatory compounds have been identified only in the female CL gland; neither has been found in the SBL gland. Thus, in contrast to the CL, which has receptor neurons which are responsive exclusively to conspecific pheromone components, the SBL has a class of receptor neurons which is responsive to a minor component of another species' pheromone blend. Field-trapping assays in which Z-5,12:AC is added to the SBL blend suggest that this single CL component is a powerful inhibitor of male SBL behavioral responses to conspecific pheromone blends. The difference observed in the specificity of the receptor neurons in this second class of sensilla are thus believed to play an integral role in the isolation processes that are maintained between these two species and may well account for the observed behavioral differences in their responses to heterospecific pheromone blends.     

Prior host feeding experience influences ovipositional but not feeding preference in a polyphagous insect herbivore

Black vine weevils, Otiorhynchus sulcatus (Fabricius) (Coleoptera: Curculionidae), are globally‐distributed polyphagous pests of many horticultural crops. We investigated how adult weevils were affected by host switching and, in particular, how host plant species nutritional and defensive chemistry affected subsequent host plant species selection and oviposition. Adults were fed one of three host plant species, blackcurrant [Ribes nigrum L. (Grossulariaceae)], raspberry [Rubus idaeus L. (Rosaceae)], or strawberry [Fragaria x ananassa Duchesne (Rosaceae)], throughout their pre‐reproductive periods and then subjected to behavioral choice assays with these plants. Foliar chemistry differed significantly among the three host plant species. Compared to raspberry and strawberry foliage, blackcurrant foliage was 13% lower in nitrogen, 3% higher in carbon, and 28% higher in phenolic compounds. Initial host plant species had a significant effect on weevil mortality, with more weevils dying when previously fed blackcurrant (12%) than strawberry (3%) or raspberry (0%) regardless of subsequent host. Initial host plant species also affected oviposition, with weevils laying only ca. two eggs per week when previously fed blackcurrant, compared to those on raspberry or strawberry (ca. 11 and 15 eggs per week, respectively). When given a choice, weevils discriminated among host plant species and tended to oviposit on plants on which they had previously fed, even when the plant was nutritionally inferior for egg production and adult survival. In contrast, feeding behavior was only affected by the current host plant species. Feeding and oviposition were related to leaf chemistry only in blackcurrant, as leaf consumption was negatively correlated with foliar carbon and zinc concentrations, and positively correlated with foliar phosphorus and potassium concentrations.     

Selective receptor neurone responses to E-β-ocimene, β-myrcene, E,E-α-farnesene and homo-farnesene in the moth Heliothis virescens, identified by gas chromatography linked to electrophysiology

An important question in olfaction is for which odorants receptor neurones have evolved. In the present study, olfactory receptor neurones on the antennae of the tobacco budworm moth Heliothis virescens were screened for sensitivity to naturally occurring plant-produced volatiles by the use of gas chromatography linked to electrophysiology. Volatiles of host as well as non-host plants collected by headspace techniques were used for stimulating the neurones, sequentially via two columns, one polar and one nonpolar installed in parallel in the gas chromatograph. Three types of neurones presented in this paper responded to one, two or three compounds for which the retention times were determined in both column types. The chemical structures of the active components were determined on the basis of mass spectrometry linked to gas chromatography, indicating E-beta-ocimene and beta-myrcene as stimulants for neurone type 1, E,E-alpha-farnesene for neurone type 2 and homo-farnesene for neurone type 3. Re-testing authentic materials verified the identifications for the type 1 neurones. The results demonstrate a high specificity for the three types of neurones by strong responses to one or two structurally similar compounds out of hundreds present in a large variety of plants. The study exemplifies plant odour detection by narrowly tuned receptor neurones in a polyphagous moth species.     

Functional characteristics of a tiny but specialized olfactory system: olfactory receptor neurons of carrot psyllids (Homoptera: Triozidae)

With only approximately 50 olfactory receptor neurons (ORNs), the carrot psyllid Trioza apicalis (Homoptera: Psylloidea) may have the smallest olfactory system described in adult Neopteran insects. Using single sensillum recordings (SSR) and gas chromatograph-linked SSR, we characterized 4 olfactory sensilla forming a distinct morphological type, which together house approximately 25% of all ORNs. We recorded responses to extracts and single constituents from Daucus carota ssp. sativus, from the conifers Picea abies, Pinus sylvestris, and Juniperus communis, as well as from male and female T. apicalis. Receptor neurons were highly selective; only 9 compounds in total elicited repeatable responses, and each neuron responded to at most 3 individual compounds. Chemical profiles of carrot and conifers showed significant overlap, with 4 out of 9 electrophysiologically active compounds occurring in more than one type of extract, but a carrot-specific compound elicited the most repeated responses. We identified 4 tentative neuron classes and found a rather high degree of neuronal redundancy, with 1 neuron class present in 3 and another present in all 4 of the sensilla, respectively.